Abstract: Triple-negative breast cancer (TNBC) is characterized by high aggressive nature, high metastatic potential, and distinct molecular characteristics from other subtypes of breast cancer. Effective targeted therapies are in urgent needs to reduce the mortality of patients with TNBC. Targeting cancer metabolic vulnerabilities is a promising therapeutic strategy for cancer therapy. We revealed that ?cyst(e)ine-addiction/dependency? is a hallmark of a subset of TNBC. Limiting cellular cysteine by targeting cystine transport and utilization induces extensive necrosis in cysteine-addictive cancer. However, most TNBC remains resistant to the targeted cysteine-addiction therapy. Our preliminary studies revealed that the potent epigenetic sensitizer tubacin (a traditional HDAC6 inhibitor) can induce synthetic lethality of cysteine-addiction in cysteine-independent cancer cells. However, tubacin-induced synthetic-lethal effects were not mediated by inhibition of endogenous HDAC6 activity, suggesting tubacin may bind new molecular targets that engaged in the synthetic- lethality. Moreover, the transcriptomic analysis revealed upregulation of genes involved in zinc homeostasis in cancer cells treated with combined erastin and tubacin treatment. We hypothesize that tubacin-promoting synthetic lethality of cysteine-addiction is mediated by increased cellular labile zinc. The objective of this proposal is to identify new molecular targets of epigenetic sensitizers, characterize the underlying synthetic-lethal mechanism, and exploit the synthetic lethality of cysteine- addiction as a novel therapeutic strategy to treat various cysteine-independent breast cancer. Specifically, we focus on three aspects: Aim 1 To identify and validate new molecular target(s) of tubacin involved in the synthetic lethality of cysteine-addiction by affinity purification and proteomics analysis. Aim 2 To determine and characterize the death-inducing mechanism of zinc in tubacin- mediated synthetic lethality. Aim 3 To validate the in vitro observations and evaluate the tumor suppressive potency of epigenetic sensitizer tubacin using mouse claudin-high TNBC xenografts. We believe that exploiting the synthetic-lethality of cysteine-addiction can be a novel therapeutic strategy to broaden the application of targeted cysteine-addiction therapy in treatment of different subtypes of breast cancer, not limit in cysteine-addictive TNBC.